Apparatus for Separating Particles Utilizing Engineered Acoustic Contrast Capture Particles

ABSTRACT

An apparatus for separating particles from a medium includes a capillary defining a flow path therein that is in fluid communication with a medium source. The medium source includes engineered acoustic contrast capture particle having a predetermined acoustic contrast. The apparatus includes a vibration generator that is operable to produce at least one acoustic field within the flow path. The acoustic field produces a force potential minima for positive acoustic contrast particles and a force potential minima for negative acoustic contrast particles in the flow path and drives the engineered acoustic contrast capture particles to either the force potential minima for positive acoustic contrast particles or the force potential minima for negative acoustic contrast particles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/295,934, filed Nov. 14, 2011, which is a continuation of U.S. patentapplication Ser. No. 11/784,928, filed Apr. 9, 2007, the contents ofwhich is incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under Contract NumberDE-AC51-06NA25396 awarded by the United States Department of Energy. TheGovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to field-based separation ofparticles in a medium utilizing acoustic pressure.

BACKGROUND

It was realized early in ultrasonic transport phenomena that particleswith different mechanical properties (density and compressibility) canbe separated in a solution. Particles in an ultrasonic standing wavefield that are more dense and less compressible than the backgroundmedium are transported to a different spatial location than particlesthat are less dense and more compressible than the background medium,producing a true binary separator based upon mechanical properties.Ultrasonic radiation pressure has been demonstrated as a viable means tomanipulate and locally trap particles in microfluidic environments. Inapplication, the acoustic radiation pressure force depends uponfrequency of excitation, pressure amplitude within the medium, and thedensity/compressibility contrast between the particle of interest andthe host medium. Within an applied ultrasonic standing wave, particlesexperience a drift force resulting from acoustic radiation pressure thattransports the particles to a position within the applied standing wavethat corresponds to minima in the acoustic radiation pressure forcepotential. In general, the location of this minima is located in adifferent spatial location for particles that are more dense and lesscompressible than the background medium in comparison to particles thatare less dense and more compressible. The technique has beensuccessfully demonstrated in particle conditioning experiments involvingtrajectory steering, agglomeration, retainment, mixing, selectiveretainment and deposition of cells on a surface.

Separation utilizing acoustic radiation pressure has not been readilyimplemented in biological problems since most biological particles ofinterest (e.g. red blood cells, white blood cells, bacteria, spores,etc.) all are more dense and less compressible than water. Recentbiological assays have implemented means to separate analytes fromsample solutions by taking advantage of their physical properties. Forinstance, positively charged silica surfaces are used to capture highlynegative charged DNA molecules from complex samples. More recently,capture particles have been employed to capture biological particles ofinterest. This has been especially true for magnetic field-basepurification methods where magnetic particles are used to captureparticles of interest from within a sample and then separated from thesample using magnetic fields.

It is desirable, therefore, to provide an apparatus and method forseparating particles within a medium utilizing engineered captureparticles and acoustic radiation pressure.

SUMMARY

An apparatus and a method for separating particles from a mediumincludes a capillary defining a flow path therein that is in fluidcommunication with a medium source. The medium source includesengineered acoustic contrast capture particle having a predeterminedacoustic contrast. The apparatus includes a vibration generator that isoperable to produce at least one acoustic field within the flow path.The acoustic field produces a force potential minima for positiveacoustic contrast particles and a force potential minima for negativeacoustic contrast particles in the flow path and drives the engineeredacoustic contrast capture particles to either the force potential minimafor positive acoustic contrast particles or the force potential minimafor negative acoustic contrast particles.

The engineered acoustic contrast capture particles may have a negativeacoustic contrast and may have a density/compressibility ratio less thanthat of the medium source. Alternatively, the engineered acousticcontrast capture particles may have a positive acoustic contrast and mayhave a density/compressibility ratio greater than that of the mediumsource.

Alternatively, the medium source includes particles having positiveacoustic contrast and particles having negative acoustic contrast andthe acoustic field drives the positive acoustic contrast particles tothe potential minima for positive acoustic contrast particles and thenegative acoustic contrast particles to the potential minima fornegative acoustic contrast particles. The positive acoustic contrastparticles may be bioparticles. The force potential minima for positivecontrast particles may be a pressure node and the force potential minimafor negative contrast particles may be a pressure antinode.

Alternatively, the engineered acoustic contrast capture particles arefunctionalized to bind to corresponding targets in the medium. Theengineered acoustic contrast capture particles may bind to the targetswith antibodies. The targets may be biological molecules or particles.The vibration generator may be a transducer or a line-drive element.

Alternatively, the acoustic field is a dipole acoustic field and theforce potential minima for positive acoustic contrast particles may belocated along a center of the flow path and the force potential minimafor negative acoustic contrast particles may be located adjacent a wallof the capillary. Alternatively, the acoustic field is an axisymmetricacoustic field and the force potential minima for negative acousticcontrast particles may be located along a center of the flow path andthe force potential minima for positive acoustic contrast particles maybe located adjacent a wall of the capillary.

Alternatively, the capillary is an inner capillary disposed within anouter capillary and the vibration generator may be disposed adjacent theouter capillary. The capillary may be quartz, glass, or plastic. Theapparatus may further comprise a laser beam for analysis of theparticles in the medium. The vibration generator may be capable ofalternately producing a dipole acoustic field and an axisymmetricacoustic field.

Objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 is a schematic of contrast factors of various materials;

FIGS. 2 a, 2 b and 2 c are schematic views of an apparatus in accordancewith the present invention shown in various stages of acoustic fieldexcitation;

FIGS. 3 a and 3 b are microscopic photographs showing bioparticles andcapture particles in a capillary of the present invention; and

FIG. 4 is a schematic view of an apparatus in accordance with thepresent invention shown with an analyzing laser.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The acoustic radiation pressure force on a compressible, sphericalparticle of volume V in an arbitrary acoustic field can be written interms of an acoustic radiation pressure force potential U:

$U = {\frac{4}{3}\pi \; {a^{3}\left\lbrack {{\left( {\beta_{o}\frac{\langle p^{2}\rangle}{2}} \right)f_{1}} - {\frac{3}{2}\left( \frac{\rho_{o}{\langle v^{2}\rangle}}{2} \right)f_{2}}} \right\rbrack}}$

Here, a is the particle radius, β_(o) is the compressibility of thesurrounding fluid, and ρ_(o) is the density of the surrounding fluid.The pressure and velocity of the acoustic field in the absence of theparticle are described by p and v, respectively, and the bracketscorrespond to a time-averaged quantity. The terms f₁ and f₂ are thecontrast terms that determine how the mechanical properties of theparticle differ from the background medium. They are given by:

$f_{1} = {1 - \frac{\beta_{p}}{\beta_{o}}}$$f_{2} = \frac{2\left( {\rho_{p} - \rho_{o}} \right)}{\left( {{2\rho_{p}} - \rho_{o}} \right)}$

The subscript p corresponds to intrinsic properties of the particle. Theforce F acting on a particle is related to the gradient of the forcepotential by:

F=−vU

-   -   Particles will be localized at positions where the potential U        displays a minima.

For acoustic wavefields comprised of plane waves, the potential minimaof U occur at either pressure nodes or antinodes depending upon thesigns of f₁ and f₂. Most particles and cells of interest in a water oraqueous background medium have properties that yield positive values off₁ and f₂. These particles are termed positive acoustic contrastparticles. Under the plane wave approximation, these particles willmigrate to the pressure nodes (velocity antinodes) in the system.Examples of these particles in water-based buffers include erythrocytes,leukocytes, bacteria, yeast, or any other particles where density andcompressibility contrast terms f₁ and f₂ are positive. Materials such asfat globules and gas bubbles yield negative values of both f₁ and f₂ andare termed negative contrast particles. Under the plane waveapproximation, these particles will move to the location of a pressureantinode. This system can be used as a binary particle separator thatspatially localizes particles into discrete locations in the wavefield(pressure nodes or antinodes) based upon positive or negative acousticcontrast of the particles relative to the background medium.

When the wavefield takes on greater complexity than described by planewave approximations (e.g. cylindrical or spherical waves), the locationof the localization of the particles within the wavefield is determinedby the minima of the force potential U. In these cases, the pressurenodes and antinodes do not generally occur at the same spatial locationsas the potential minima. But, similar to the plane wave case, theparticles can generally be separated in a binary manner based upon theirintrinsic properties relative to the background medium. The binaryspatial positions where positive and negative acoustic contrastparticles localize is now determined by minima in the force potential U.

The contrast factors f₁ and f₂ demonstrate the significance of therelative values of the intrinsic properties of the particles and themedium. As shown in FIG. 1, for most particles in bio-research andbiomedical applications including, but not limited to, erythrocytes,phagocytes, spores, yeast, fungi, and bacteria, as well as plastics,sand, common sediments, and metals, the contrast term is positive forparticles in an aqueous background and all particles in a given sampleare forced to the location of the same potential minima in the standingwave field. Therefore, acoustic binary separation of most biologicalparticles in an aqueous solution is not feasible without adjusting theproperties of the background medium. Separation of lipids from blood byusing their negative acoustic contrast relative to the other bloodcomponents has been demonstrated by others.

The engineered acoustic contrast capture particles, when attached tobioparticles of interest, shifts the effective density andcompressibility of the bioparticle/capture particle construct frompositive to negative or negative to positive depending upon thepreferred separation criteria relative to other particles in the system.As an example, an engineered capture particle is designed with lowdensity and high compressibility relative to an aqueous medium. Theparticle is functionalized with anit-CD45 ligands and introduced into ablood sample to bind to CD45 markers on leukocytes. Under conditionswhere the blood sample is suspended in an aqueous buffer, both theerythrocytes and leukocytes have positive acoustic contrast and underthe action of an acoustic standing wave, are forced to the samepotential minima By attaching an engineered particle with negativeacoustic contrast to the leukocyte, the capture particle/leukocyteconstruct takes on negative acoustic contrast and be advantageouslyseparated from the positive contrast population of erythrocytes.

Referring now to FIGS. 2 a-2 c, apparatus in accordance with the presentinvention is indicated generally at 10. The apparatus 10 includescapillary 12 defining therein flow path or stream 14 in fluidcommunication with a medium source (not shown) within walls 16 ofcapillary 12. Capillary 12 is preferably constructed from quartz, glass,plastic, or any suitable material. Preferably, capillary 12 is circularin cross-section but those skilled in the art will appreciate that thecross-section of capillary 12 may be of any suitable geometry including,but not limited to, elliptical, square, rectangular or the like. Themedium source may supply water, or any suitable liquid to flow path orstream 14, as will be appreciated by those skilled in the art. Capillary12 is fully disposed within outer capillary 18. Vibration generator 20,such as a transducer the like, is disposed adjacent or mounted to anexterior surface of capillary 18 and is capable of producing at leastone acoustic or displacement field within flow stream 14 of capillary12. Alternatively, vibration generator 20 is a line-drive element, adisplacement generator, or any other type of vibration generatoradjacent or mounted to the exterior surface of capillary 12 capable ofproducing at least one acoustic or displacement field within flow pathor stream 14 including, but not limited to, a contact aperture, a linecontact, and a full encompassment of capillary 12, as will beappreciated by those skilled in the art. Under different symmetryexcitations of the displacement field, the position of a force potentialminima for positive contrast particles can be located along the centralaxis or flow stream 14 of capillary 12 or the force potential minima forpositive contrast particles can be located at a position along wall 16.In these situations, the force potential minima for negative acousticcontrast particles will be located at the position not occupied bypositive acoustic contrast particles (e.g. positive acoustic contrastparticles along the central axis of flow stream 14 corresponds tonegative acoustic contrast particles at a position along capillary wall16 or negative acoustic contrast particles along the central axis offlow stream 14 corresponds to positive acoustic contrast particles at aposition along capillary wall 16).

Disposed within flow stream 14 are bioparticles 22 and engineeredacoustic contrast capture particles 24. As shown in FIG. 2 a, with noacoustic field being generated by vibration generator 20 (i.e. theacoustic field is inactive), bioparticles 22 and negative contrastcapture particles 24 are homogeneously distributed within flow stream 14of capillary 12. As shown in FIG. 2 b, when a dipole acoustic excitationand resulting dipole acoustic field is implemented by vibrationgenerator 20, bioparticles 22 are focused to the center of flow stream14 of capillary 12 (force potential minima for positive acousticcontrast particles) and engineered acoustic contrast capture particleswith negative contrast 24 are transported to capillary wall 16 (forcepotential minima for negative acoustic contrast particles). Conversely,as shown in FIG. 2 c, when an axisymmetric excitation and resultingaxisymmetric acoustic field is implemented by vibration generator 20,all bioparticles 22 are sent to capillary wall 16 (force potentialminima for positive acoustic contrast particles) and engineered acousticcontrast capture particles with negative contrast 24 are focused to thecentral axis of flow stream 14 of capillary 12 (force potential minimafor negative acoustic contrast particles).

In a non-limiting example, background bioparticles 22 comprising 6 .mu.mlatex spheres and engineered acoustic contrast capture particles withnegative contrast 24 comprising hollow, glass microspheres having lowdensity, high compressibility characteristics were utilized in apparatus10. Upon activation of vibration generator 20 generating a dipoleacoustic field, such as shown in FIG. 2 b, latex spheres 22 are focusedto the center of flow stream 14 of capillary 12 while hollow glassspheres 24 are transported to wall 16 of capillary 12. This is shown inFIGS. 3 a and 3 b. A microscope with a narrow depth of field (not shown)is utilized to demonstrate the location of positive and negativecontrast particles in apparatus 10. In FIG. 3 a, the focal plane of themicroscope intersects the central axis of flow stream 14 of capillary 12where positive contrast particles 22 (latex spheres) have been focusedalong the axis and engineered negative contrast particles (glassmicrospheres) can be seen along capillary wall 16. In FIG. 3 b, thefocal plane of the microscope intersects an uppermost boundary of innercapillary wall 16. Here, negative contrast particles 24 (glassmicrospheres) are located adjacent capillary wall 16. Before activationof the acoustic field, particles 22 and 24 were homogenously distributedthroughout capillary 12, such as shown in FIG. 2 a.

Referring now to FIG. 4, in this embodiment, engineered acousticcontrast capture particles with negative contrast 24′ are functionalizedwith a specific coating to bind a target particle of interest, such asby functionalizing and coating engineered negative contrast captureparticles 24′ with antibody 26. Negative contrast capture particles 24′and antibodies 26 are mixed with a sample, incubated, and are bound totarget cells 28. Target cells 28 are preferably biological molecules orparticles. Upon flowing the sample through flow stream 14 of capillary12 excited in an axisymmetric vibration mode by vibration generator 20,engineered negative contrast capture particles 24′ with captured targetcells 28 are focused to the center of flow stream 14 of capillary 12 foranalysis by laser beam 30 (flow cytometry) or separation. The samplebackground particles (as they are bioparticles similar to bioparticles22 shown in FIGS. 2 a-2 c) are forced to wall 16 of capillary 12. In aflow cytometer (not shown), laser beam 30 is focused to probe only thoseparticles that transport along the axis of flow stream 14 of capillary12.

Apparatus 10 preferably includes vibration generator 20 capable ofdriving capillary 12 in both axisymmetric and dipole modes,advantageously allowing for separate analysis of target particles 28 andremaining sample background particles 22. Engineered negative acousticcontrast capture particles 24 are transported to the position of aminima of the force potential for negative contrast particles in anacoustic field. Conversely, all other bioparticles 22 are transported tothe position of a minima of the force potential for positive contrastparticles in an acoustic field. In a flow cytometer, functionalized,engineered negative contrast capture particles 24′ can be mixed with asample. The untrapped sample or specific binding to capture particles24′ can be analyzed by placing or designing either the force potentialminima for positive acoustic contrast particles or negative acousticcontrast particles at the center of the sample analysis flow stream 14.

Apparatus 10 advantageously takes advantage of the fact that mostbioparticles, such as bioparticles 22 shown in FIGS. 2 a through FIG. 4,possess positive acoustic contrast and can be spatially isolated at aspecific predetermined location defined by the force potential minimafor positive acoustic contrast particles in an acoustic field, as theonly particles that have negative contrast are acoustically similar tolipids/fats. By engineering capture particles 24 with a negativeacoustic contrast, it is possible to separate engineered negativecontrast capture particles 24 and 24′ from biological particles 22 (andmost other background particles 22) in a sample, as shown in FIG. 4.Under the action of an acoustic field, biological particles 22 willtransport to the minima in the force potential for positive contrastparticle, while engineered negative contrast capture particles 24 willtransport to the minima in the force potential for negative contrastparticles. In general, the spatial location defined by the forcepotential minima of positive and negative acoustic contrast particlesare isolated from one another or at distinct predetermined locationswithin flow stream 14 of capillary 12.

Apparatus 10 advantageously employs engineered acoustic contrast captureparticles with negative contrast 24 and 24′ having adensity/compressibility ratio less than that of the medium source (i.e.particles 24 and 24′ are less dense and more compressible than thebackground fluid medium in the medium source, such as water). If themedium in flow stream 14 of capillary 12 is not water, the contrastproperties of engineered acoustic contrast capture particles 24 and 24′may be advantageously adjusted according to the medium to take on eithernegative or positive contrast values. Engineered acoustic contrastcapture particles 24 and 24′ are used to capture bioparticles ofinterest 28 in a sample. When used in conjunction with an acousticallydriven cylindrical channel, engineered acoustic contrast captureparticles 24 and 24′ can be forced to the center of flow stream 14 orwall 16 of capillary 12 and separated from the remaining sampleconstituents. This is very effective in new assays for flow cytometrythat require target cells or constituents to be separated from abackground. Alternatively, engineered acoustic contrast particles 24 and24′ have a positive acoustic contrast and have a density/compressibilityratio greater than that of the medium source (i.e. particles 24 and 24′are more dense and less compressible than the background fluid medium inthe medium source, such as water).

Apparatus 10 may be advantageously utilized in commercial applicationsincluding, but not limited to, applications involving the separation ofspecific targets in biological samples.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverin the appended claims all such modifications and equivalents. Theentire disclosures of all references, applications, patents, andpublications cited above and/or in the attachments, and of thecorresponding application(s), are hereby incorporated by reference.

What is claimed:
 1. An apparatus for separating particles from a medium,comprising: a capillary defining a flow path therein, said flow path influid communication with a medium source, said medium source includingengineered acoustic contrast capture particles, at least some of theengineered acoustic contrast capture particles being configured to bindto a predetermined bioparticle and having an acoustic contrast thatdiffers from the acoustic contrast of the predetermined bioparticle; anda vibration generator configured to produce at least one acoustic fieldwithin said flow path, said at least one acoustic field being configuredto drive said engineered acoustic contrast capture particles to one ormore a force potential minima.
 2. The apparatus of claim 1, wherein theat least some of the engineered acoustic contrast capture particles havea negative acoustic contrast and wherein the predetermined bioparticlehas a positive acoustic contrast.
 3. The apparatus of claim 2, whereinsaid engineered acoustic contrast particles have adensity/compressibility ratio less than that of the medium source. 4.The apparatus of claim 1, wherein said engineered acoustic contrastcapture particles have a positive acoustic contrast.
 5. The apparatus ofclaim 4, wherein said engineered acoustic contrast particles have adensity/compressibility ratio greater than that of the medium source. 6.The apparatus of claim 1, wherein said one or more force potentialminima is a pressure node or a pressure antinode.
 7. The apparatus ofclaim 1, wherein said engineered acoustic contrast capture particlesbind via antibodies to said bioparticles.
 8. The apparatus of claim 7,wherein said bioparticles are biological molecules.
 9. The apparatus ofclaim 1, wherein said vibration generator is a transducer.
 10. Theapparatus of claim 1, wherein said vibration generator is a line-driveelement.
 11. The apparatus of claim 1, wherein said at least oneacoustic field is a dipole acoustic field.
 12. The apparatus of claim 1,wherein said at least one acoustic field is an axisymmetric acousticfield.
 13. The apparatus of claim 1, wherein said capillary is an innercapillary disposed within an outer capillary, said vibration generatorbeing disposed adjacent said outer capillary.
 14. The apparatus of claim1, further comprising a laser beam for analysis of said particles insaid medium.
 15. The apparatus of claim 1, wherein said vibrationgenerator is capable of alternately producing a dipole acoustic fieldand an axisymmetric acoustic field.
 16. A system comprising: a capillarydefining a flow path therein, a fluid medium source coupled to thecapillary, the fluid medium source configured to supply a fluid mediumto the capillary; a vibration generator coupled to the capillary; and aplurality of engineered positive acoustic contrast capture particlesdisposed in the fluid medium, the engineered positive acoustic contrastcapture particles having a higher density/compressibility ratio than thedensity/compressibility ratio of the fluid medium.
 17. The system ofclaim 16, wherein the vibration generator is configured to produce anacoustic field within the flow path of the capillary.
 18. The system ofclaim 16, wherein the vibration generator is configured to produce aforce potential minima in the fluid medium, the force potential minimadriving the engineered negative acoustic contrast capture particles tothe minima.
 19. The system of claim 16, wherein the engineered acousticcontrast capture particles are configured to bind to a predeterminedbioparticle.
 20. The system of claim 19, wherein the engineered acousticcontrast capture particles have an acoustic contrast that differs fromthe acoustic contrast of the predetermined bioparticle.